ASSOCIATIONS BETWEEN ERYTHROCYTE BAND-3 PROTEIN AND ALDOLASE IN DETERGENT SOLUTION - DETERMINING THEIR STOICHIOMETRY BY ANALYTICAL ULTRACENTRIFUGATION

The cytoplasmic domain of band 3, the predominant polypeptide of the e rythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oli gomers as Ligand binding sites. The experiments were performed on mixt ures of solubilized band 3 and aldolase in solutions of a nonionic det ergent, nonaethyleneglycol lauryl ether. The main technique applied wa s sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To f acilitate the evaluations, the aldolase was labelled with a dye. The f ollowing results were obtained. (1) With unmodified band 3, aldolase i s bound exclusively or at least predominantly to the band 3 tetramer ( but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 d imers (stabilized either covalently or noncovalently) can also associa te with aldolase and can bind up to two aldolase tetramers. The result s described, together with those reported previously, point at a promi nent role of the band 3 tetramer in ligand binding.